Diagnostic value of increased sensitivity by massively parallel sequencing

Routine molecular diagnostics modalities are unable to confidently detect low frequency mutations (<5-15%) that may indicate response to targeted therapies. We confirm the presence of a low frequency NRAS mutation in a rectal cancer patient using massively parallel sequencing when previous Sanger sequencing results proved negative and Q-PCR testing inconclusive. There is increasing evidence that these low frequency mutations may confer resistance to anti-EGFR therapy. In view of negative/inconclusive Sanger sequencing and Q-PCR results for NRAS mutations in a KRAS wt rectal case, the diagnostic biopsy and 4 distinct subpopulations of cells in the resection specimen after conventional chemo/radiotherapy were massively parallel sequenced using the Ion Torrent PGM. DNA was derived from FFPE rectal cancer tissue and amplicons produced using the Cancer Hotspot Panel V2 and sequenced using semiconductor technology. NRAS mutations were observed at varying frequencies in the patient biopsy (12.2%) and all four subpopulations of cells in the resection with an average frequency of 7.3% (lowest 2.6%). The results of the NGS also provided the mutational status of 49 other genes that may have prognostic or predictive value, including KRAS and PIK3CA. NGS technology has been postulated in diagnostics because of its capability to generate results in large panels of clinically meaningful genes in a cost-effective manner. This case illustrates another potential advantage of this technology: its use for detecting low frequency mutations that may influence therapeutic decisions in cancer treatment.

Challenging the cancer molecular stratification dogma: Intratumoral heterogeneity undermines consensus molecular subtypes and potential diagnostic value in colorectal cancer

Purpose:
A number of independent gene expression profiling studies have identified transcriptional subtypes in colorectal cancer (CRC) with potential diagnostic utility, culminating in publication of a CRC Consensus Molecular Subtype classification. The worst prognostic subtype has been defined by genes associated with stem-like biology. Recently, it has been shown that the majority of genes associated with this poor prognostic group are stromal-derived. We investigated the potential for tumor misclassification into multiple diagnostic subgroups based on tumoral region sampled.

Experimental Design:
We performed multi-region tissue RNA extraction/transcriptomic analysis using Colorectal Specific Arrays on invasive front, central tumor and lymph node regions selected from tissue samples from 25 CRC patients.

Results:
We identified a consensus 30 gene list which represents the intratumoral heterogeneity within a cohort of primary CRC tumors. Using a series of online datasets, we showed that this gene list displays prognostic potential (HR=2.914 (CI 0.9286-9.162) in stage II/III CRC patients, but in addition we demonstrated that these genes are stromal derived, challenging the assumption that poor prognosis tumors with stem-like biology have undergone a widespread Epithelial Mesenchymal Transition (EMT). Most importantly, we showed that patients can be simultaneously classified into multiple diagnostically relevant subgroups based purely on the tumoral region analysed.

Conclusions:
Gene expression profiles derived from the non-malignant stromal region can influence assignment of CRC transcriptional subtypes, questioning the current molecular classification dogma and highlighting the need to consider pathology sampling region and degree of stromal infiltration when employing transcription-based classifiers to underpin clinical decision-making in CRC.

Diagnostic value of surgical lung biopsy: comparison with clinical and radiological diagnosis

OBJECTIVE: To determine overall and disease-related accuracy of the clinical/imagiological evaluation for pulmonary infiltrates of unknown aetiology, compared with the pathological result of the surgical lung biopsy (SLB) and to evaluate the need for the latter in this setting. METHODS: We conducted a retrospective review of the experiences of SLB in 366 consecutive patients during the past 5 years. The presumptive diagnosis was based on clinical, imagiological and non-invasive or minimally invasive diagnostic procedures and compared with the gold standard of histological diagnosis by SLB. We considered five major pathological groups: diffuse parenchymal lung disease (DPLD), primitive neoplasms, metastases, infectious disease and other lesions. Patients with previous histological diagnosis were excluded. RESULTS: In 56.0% of patients (n=205) clinical evaluation reached a correct diagnosis, in 42.6% a new diagnosis was established (n=156) by the SLB, which was inconclusive in 1.4% (n=5). The pre-test probability for each disease was 85% for DPLD, 75% for infectious disease, 64% for primitive neoplasms and 60% for metastases. Overall sensitivity, specificity, positive and negative predictive values for the clinical/radiological diagnosis were 70%, 90%, 62% and 92%, respectively. For DPLD: 67%, 90%, 76% and 85%; primitive neoplasms: 47%, 90%, 46% and 90%; metastases: 99%, 79%, 60% and 99%; infectious disease 38%, 98%, 53% and 96%. CONCLUSIONS: Despite a high sensitivity and specificity of the clinical and imagiological diagnosis, the positive predictive value was low, particularly in the malignancy group. SLB should be performed in pulmonary infiltrates of unknown aetiology because the clinical/imagiological assessment missed and/or misdiagnosed an important number of patients.

Diagnostic value of H3F3A mutations in giant cell tumour of bone compared to osteoclast-rich mimics

Driver mutations in the two histone 3.3 (H3.3) genes, H3F3A and H3F3B, were recently identified by whole genome sequencing in 95% of chondroblastoma (CB) and by targeted gene sequencing in 92% of giant cell tumour of bone (GCT). Given the high prevalence of these driver mutations, it may be possible to utilise these alterations as diagnostic adjuncts in clinical practice. Here, we explored the spectrum of H3.3 mutations in a wide range and large number of bone tumours (n 5 412) to determine if these alterations could be used to distinguish GCT from other osteoclast-rich tumours such as aneurysmal bone cyst, nonossifying fibroma, giant cell granuloma, and osteoclast-rich malignant bone tumours and others. In addition, we explored the driver landscape of GCT through whole genome, exome and targeted sequencing (14 gene panel). We found that H3.3 mutations, namely mutations of glycine 34 in H3F3A, occur in 96% of GCT. We did not find additional driver mutations in GCT, including mutations in IDH1, IDH2, USP6, TP53. The genomes of GCT exhibited few somatic mutations, akin to the picture seen in CB. Overall our observations suggest that the presence of H3F3A p.Gly34 mutations does not entirely exclude malignancy in osteoclast-rich tumours. However, H3F3A p.Gly34 mutations appear to be an almost essential feature of GCT that will aid pathological evaluation of bone tumours, especially when confronted with small needle core biopsies. In the absence of H3F3A p.Gly34 mutations, a diagnosis of GCT should be made with caution.

Eosinophilie sanguine: quel bilan, quel cheminement diagnostique [Peripheral blood eosinophilia: diagnostic value and further assessment].

Although not specific, an increased in peripheral blood eosinophils may contribute substantially to the diagnosis of numerous infectious, allergic and inflammatory diseases. The scope of this article is to detail pathologies associated with peripheral eosinophilia by order of frequency and to guide further investigations.

Diagnostic value of the Dominic Interactive Assessment with children exposed to intimate partner violence

Resumen tomado de la publicaci??n

The diagnostic value of Gram stain for initial identification of the etiologic agent of peritonitis in CAPD patients

Objective: To evaluate the effectiveness of the Gram stain in the initial diagnosis of the etiologic agent of peritonitis in continuous ambulatory peritoneal dialysis (CAPD). Design: Retrospective study analyzing the sensitivity (S), specificity (SS), positive predictive value (+PV), and negative predictive value (-PV) of the Gram stain relating to the results of cultures in 149 episodes of peritonitis in CAPD. The data were analyzed in two studies. In the first, only the cases with detection of a single agent by Gram stain were taken (Study 1). In the second, only the cases with two agents in Gram stain were evaluated (Study 2). Setting: Dialysis Unit and Laboratory of Microbiology of a tertiary medical center. Patients: Sixty-three patients on regular CAPD who presented one or more episodes of peritonitis from May 1992 to May 1995. Results: The positivity of Gram stain was 93.2% and the sensitivity was 95.7%. The values of S, SS, +PV, and -PV were respectively: 94.9%, 53.5%, 68.3%, and 90.9% for gram-positive cocci and 83.3%, 98.8%, 95.2%, and 95.6% for gram-negative bacilli. The association of gram-positive cocci plus gram-negative bacilli were predictive of growth of both in 6.8%, growth of gram-positive cocci in 13.7%, and growth of gram-negative bacilli in 72.5%. Conclusions: The Gram stain is a method of great value in the initial diagnosis of the etiologic agent of peritonitis in CAPD, especially for gram-negative bacilli.

Diagnostic value of serum activin A and follistatin levels in women with peritoneal, ovarian and deep infiltrating endometriosis

Activin A is a growth factor, produced by the endometrium, whose actions are modulated by the binding protein follistatin. Both proteins are detectable in the peripheral serum and their concentrations may be increased in women with endometriosis. The present study was designed to evaluate whether serum levels of activin A and follistatin are altered, and therefore have a potential diagnostic value, in women with peritoneal, ovarian and deep infiltrating endometriosis. We performed a multicenter controlled study evaluating simultaneously serum activin A and follistatin concentrations in women with and without endometriosis. Women with endometriosis (n 139) were subdivided into three groups: peritoneal endometriosis (n 28); ovarian endometrioma (n 61) and deep infiltrating endometriosis (n 50). The control group (n 75) consisted of healthy women with regular menstrual cycles. Blood samples were collected from a peripheral vein and assayed for activin A and follistatin using commercially available enzyme immunoassay kits. The ovarian endometrioma group had serum activin A levels significantly higher than healthy controls (0.22 0.01 ng/ml versus 0.17 0.01 ng/ml, P 0.01). None of the endometriosis groups had serum follistatin levels which were significantly altered compared with healthy controls; however, levels found in the endometrioma group (2.34 0.32 ng/ml) were higher than that in the deep endometriosis group (1.50 0.17 ng/ml, P 0.05). The area under the receiver operating characteristic curve of activin A was 0.700 (95 confidence interval: 0.6050.794), while that of follistatin was 0.620 (95 confidence interval: 0.5100.730) for the diagnosis of ovarian endometrioma. The combination of both markers into a duo marker index did not improve significantly their diagnostic accuracy. The present study demonstrated that serum activin A and follistatin are not significantly altered in peritoneal or deep infiltrating endometriosis and have limited diagnostic accuracy in the diagnosis of ovarian endometrioma.

What is the diagnostic value of ultrasonography compared to physical evaluation in patients with idiopathic carpal tunnel syndrome?

[EN] OBJECTIVE: Our hypothesis is that sonography performed by the rheumatologist in patients with suspected carpal tunnel syndrome (CTS) has higher diagnostic value compared to physical evaluation. METHODS: Adult patients with suspected idiopathic CTS, defined by sensory symptoms over the distribution of the median nerve with or without positive results with the Phalen and/or the Tinel's maneuvers were included. The diagnosis of CTS was indicated by typical symptoms daily for at least 3 months and a positive nerve conduction study. One rheumatologist unaware of the clinical and electrodiagnostic results performed an ultrasound examination of the median nerve for the area ranging from the inlet to the outlet of the carpal tunnel. Mean cross-sectional area at each level, flattening ratio and bowing of flexor retinaculum were obtained. RESULTS: Sixty-eight patients with 105 affected wrists were examined. Tinel's and Phalen's signs had a closer sensitivity (73% and 67% respectively) and specificity (40% and 30% respectively). The best swelling nerve cut-off by sonography was 9.7 mm2 at the tunnel inlet, with a sensitivity of 86%, a specificity of 48% and accuracy of 77%. A 100% positive predictive value was reached with a cross-sectional area of 13 mm2, involving 33 hands (31% of the whole sample). Maximal cross sectional area and the measurement of flexor retinaculum had an accuracy of 72% and 73% respectively. Combination of physical maneuvers and sonography not yielded more accuracy than cross-sectional area itself. CONCLUSION: In patients with clinical history of idiopathic CTS and positive nerve conduction study, sonography performed by the rheumatologist has higher diagnostic value than physical maneuvers.

Can molecular markers stratify the diagnostic value of high-grade prostatic intraepithelial neoplasia?

The diagnostic performance of isolated high-grade prostatic intraepithelial neoplasia in prostatic biopsies has recently been questioned, and molecular analysis of high-grade prostatic intraepithelial neoplasia has been proposed for improved prediction of prostate cancer. Here, we retrospectively studied the value of isolated high-grade prostatic intraepithelial neoplasia and the immunohistochemical markers ?-methylacyl coenzyme A racemase, Bcl-2, annexin II, and Ki-67 for better risk stratification of high-grade prostatic intraepithelial neoplasia in our local Swiss population. From an initial 165 diagnoses of isolated high-grade prostatic intraepithelial neoplasia, we refuted 61 (37%) after consensus expert review. We used 30 reviewed high-grade prostatic intraepithelial neoplasia cases with simultaneous biopsy prostate cancer as positive controls. Rebiopsies were performed in 66 patients with isolated high-grade prostatic intraepithelial neoplasia, and the median time interval between initial and repeat biopsy was 3 months. Twenty (30%) of the rebiopsies were positive for prostate cancer, and 10 (15%) showed persistent isolated high-grade prostatic intraepithelial neoplasia. Another 2 (3%) of the 66 patients were diagnosed with prostate cancer in a second rebiopsy. Mean prostate-specific antigen serum levels did not significantly differ between the 22 patients with prostate cancer and the 44 without prostate cancer in rebiopsies, and the 30 positive control patients, respectively (median values, 8.1, 7.7, and 8.8 ng/mL). None of the immunohistochemical markers, including ?-methylacyl coenzyme A racemase, Bcl-2, annexin II, and Ki-67, revealed a statistically significant association with the risk of prostate cancer in repeat biopsies. Taken together, the 33% risk of being diagnosed with prostate cancer after a diagnosis of high-grade prostatic intraepithelial neoplasia justifies rebiopsy, at least in our not systematically prostate-specific antigen-screened population. There is not enough evidence that immunohistochemical markers can reproducibly stratify the risk of prostate cancer after a diagnosis of isolated high-grade prostatic intraepithelial neoplasia.

MR defecography in patients with dyssynergic defecation: spectrum of imaging findings and diagnostic value

We describe the spectrum of findings and the diagnostic value of MR defecography in patients referred with suspicion of dyssynergic defecation.

Diagnostic value of animal-side antibody assays for rapid detection of Mycobacterium bovis or Mycobacterium microti infection in South American camelids

Tuberculosis (TB) in South American camelids (SAC) is caused by Mycobacterium bovis or Mycobacterium microti. Two serological methods, rapid testing (RT) and the dual-path platform (DPP) assay, were evaluated using naturally infected SAC. The study population included 156 alpacas and 175 llamas in Great Britain, Switzerland, and the United States. TB due to M. bovis (n = 44) or M. microti (n = 8) in 35 alpacas and 17 llamas was diagnosed by gross pathology examination and culture. Control animals were from herds with no TB history. The RT and the DPP assay showed sensitivities of 71% and 74%, respectively, for alpacas, while the sensitivity for llamas was 77% for both assays. The specificity of the DPP assay (98%) was higher than that of RT (94%) for llamas; the specificities of the two assays were identical (98%) for alpacas. When the two antibody tests were combined, the parallel-testing interpretation (applied when either assay produced a positive result) enhanced the sensitivities of antibody detection to 89% for alpacas and 88% for llamas but at the cost of lower specificities (97% and 93%, respectively), whereas the serial-testing interpretation (applied when both assays produced a positive result) maximized the specificity to 100% for both SAC species, although the sensitivities were 57% for alpacas and 65% for llamas. Over 95% of the animals with evidence of TB failed to produce skin test reactions, thus confirming concerns about the validity of this method for testing SAC. The findings suggest that serological assays may offer a more accurate and practical alternative for antemortem detection of camelid TB.